Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA

• Published in: Virus research 2004-05, Vol.101 (2), p.147-153
• Main Authors: Plattet, Philippe, Zweifel, Christine, Wiederkehr, Christa, Belloy, Luc, Cherpillod, Pascal, Zurbriggen, Andreas, Wittek, Riccardo
• Format: Article
• Language: English
• Subjects: Adaptation, Physiological; Additional transcription unit; Amino Acid Substitution; Animals; Canine distemper virus; Cell Line; Cercopithecus aethiops; Cloning, Molecular; Complete nucleotide sequence; Distemper Virus, Canine - genetics; Distemper Virus, Canine - growth & development; Dogs; Gene Order; Genes, Reporter; Genes, Viral; Genome, Viral; Genomics; Green Fluorescent Proteins; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mutation, Missense; Persistent infection; Recombination, Genetic; Reverse genetics; Vero cell-adaptation; Vero Cells; Viral Proteins - chemistry; Viral Proteins - genetics
• ISSN: 0168-1702; 1872-7492
• DOI: 10.1016/j.virusres.2004.01.002

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3′ proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.